CN110244065A - The Microfluidic Immunoassay Chip and its preparation method and application of 1 albumen of progesterone receptor membrane component in a kind of detection blood - Google Patents
The Microfluidic Immunoassay Chip and its preparation method and application of 1 albumen of progesterone receptor membrane component in a kind of detection blood Download PDFInfo
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Abstract
The present invention relates to the Microfluidic Immunoassay Chips and its preparation method and application of 1 albumen PGRMC1 of progesterone receptor membrane component in detection blood a kind of；This Microfluidic Immunoassay Chip is made of chip and solid phase reaction substrate and the specific antigen PGRMC1 being coated in solid phase reaction substrate；Chip is the laminated structure with parallel microchannel, and the specific antigen PGRMC1 in solid phase reaction substrate is parallel streak-like planar structure, is mutually perpendicular to the chip microchannel, and intersecting angle is 90 °；PGRMC1 is the potential marker of possibility of breast cancer related to progestational hormone, by the content of the PGRMC1 in detection whole blood, can provide possibility for early diagnosing mammary cancer；The detection method combines Microfluidic Immunoassay Chip and chemiluminescence, it is small the quick of Microfluidic Immunoassay Chip, efficient and amount of samples have been had both, the advantages that chemiluminescence specificity is good, easy to operate, at low cost, high-throughput, the early diagnosis application prospect with breast cancer.
Description
Technical field
The present invention relates to bioassay technique fields, and in particular to 1 egg of progesterone receptor membrane component in a kind of detection blood
White Microfluidic Immunoassay Chip and its preparation method and application.
Background technique
Building High Sensitive Analysis method is always one of the target that analytical chemistry and life science are pursued.High Sensitive Analysis side
The development and its application in fields such as clinical diagnosis, environmental monitoring, food safety, bio-imagings that method has pushed analytical chemistry.
In the early diagnosis of the diseases such as serious infectious diseases, bacterium or virus infection, cancer and food and Environmental Trace noxious material
Screening in, High Sensitive Analysis method is particularly important.Immune biomarker analysis based on antibody-antigene specific recognition
Method is a kind of combined analysis method of the high degree of specificity by the high sensitivity of biomarker technology and immune response,
Have many advantages, such as that high sensitivity, specificity are good, analysis is quick.Enzyme linked immunological is representative therein, this method have high specificity,
It is reproducible, easy to operate, at low cost, be easy to be commercialized and automate the advantages that.But traditional enzyme-linked immune analytic method one
As be all based on enzymatic signal amplification between immune labeled enzyme and substrate, sensitivity can only achieve ng/mL.Meanwhile
The enzyme-linked immune analytic method reaction time is long, large labor intensity.In the analysis field for needing highly sensitive, clinical quick diagnosis,
Traditional enzyme-linked immune analytic method is difficult to meet needs, and therefore, simple and convenient quick high-sensitivity high-flux skill is established in research
Art method is vital.
In global female group, breast cancer has become one of most common invasive cancer, and women suffers from life
The probability of breast cancer is 1/8.Chinese tumour neopathy number of cases is 429.2 ten thousand within 2015, and wherein women with breast cancer number of the infected is
26.86 ten thousand, it accounts for women and newly swells the 15% of tumor, wherein 45~59 years old disease incidence is 23%.This age level is exactly female
Property encloses menopause or menopausal stage.Hormone therapy is current treatment menopause-related symptoms most efficient method, it is to maintenance menopause
Body of women health, improving the quality of living has the function of that other any single medicines cannot replace.But there is research to recognize at present
For hormone therapy can be such that breast cancer occurrence risk increases.Women treated by 5 years or more female/progestin combinations, breast cancer wind
Danger will increase (HR=1.24,95%CI:1.01~1.54).In addition, also there is most clinical studies show, hormone therapy is super
5 years are spent, the onset risk of breast cancer increases.Studies have found that the probability in healthy women mammary gland there are malignant cell is 40%,
Even if some women endogenous estrogen, progestational hormone stimulation under, mammary gland malignant cell may also can occur quickly to increase
Giving birth to and developing is clinical breast cancer.As can early detection estrogen, progestational hormone correlation mammary cancer risk, filter out high-risk breast cancer
Patient simultaneously takes corresponding measure, has great importance to the mammary cancer risk for reducing hormone therapy.But up to now, face
Lack certain susceptibility, specificity about mammary cancer risk serologic marker object on bed.In the nearest more than ten years, it was found that one
A little new progesterone receptor membrane components, wherein (the progesterone receptor membrane of progesterone receptor membrane component 1
Components 1, PGRMC1), estrogen, progestin combinations treatment can be obviously promoted the positive breast cancer of PGRMC1 expression
The hyperplasia of cell, in vitro study show, external estrogen, progestational hormone are to the breast cancer of MCF7/PGRMC1 and untransfected PGRMC1
Cell (MCF7) proliferative effect has significantly different, and estrogen and certain progestational hormone significantly increase the breast cancer for having transfected PGRMC1
The hyperplasia of cell, and tumour may be taken part in.In recent years successively about PGRMC1 in solid tumor expression report
Road, but result of study is inconsistent, while these clinical datas are more scattered, and research sample size is small, and overwhelming majority research concentrates on
Assess specific eucaryotic cell structure in breast cancer tissue and can not the expression to PGRMC1 in blood samples of patients carry out testing and evaluation, and
The latter is very necessary for the normal risk screening before hormone therapy.
In conclusion it is necessary to screen whether determining PGRMC1 is a kind of new, suitable breast cancer tumour mark for this field
Remember object, while necessary research and development detection sensitivity is high, detection is more simple and convenient, high-throughput, is suitable for rapid field
The immune detection of detection analyzes new technologies.
Summary of the invention
The object of the present invention is to provide a kind of Microfluidic Immunoassays of 1 albumen of progesterone receptor membrane component in detection blood
Chip and its preparation method and application.Current immunoassay method is solved to read in detection sensitivity, analysis speed and signal
Mode etc. is not able to satisfy the case where practical application request.There is immunoassay method of the invention high sensitivity, signal to read
Mode is simple out, the detection range of linearity is wide, analysis speed is fast, testing cost is low, the advantages such as high-throughput detection may be implemented.
It is of the present invention it is a kind of detection blood in 1 albumen of progesterone receptor membrane component Microfluidic Immunoassay Chip and
Preparation method and application the technical solution adopted is as follows:
The Microfluidic Immunoassay Chip of 1 albumen of progesterone receptor membrane component in a kind of detection blood of the present invention,
It is made of chip and solid phase reaction substrate and the specific antigen PGRMC1 being coated in solid phase reaction substrate；
Chip of the present invention is manually overmolded or the laminated structure with parallel microchannel being molded；It is excellent
Choosing, the chip have the parallel microchannel of 3-21 item；The microchannel length is 3-5cm, the microchannel diameter
500-600 μm 500-600 μm of height, the spacing of the microchannel is 1-3mm；It is described to be coated in solid phase reaction substrate
PGRMC1 is parallel streak-like planar structure, and length 3-5cm, spacing 1-3mm are mutually perpendicular to the chip microchannel,
Intersecting angle is 90 °.
The Microfluidic Immunoassay Chip of 1 albumen of progesterone receptor membrane component in a kind of detection blood of the present invention
Preparation method:
Closed microchannel is formed after chip is bonded with solid phase reaction substrate, is injected separately into Xiang Suoshu microchannel
20uL, concentration range are 3ug/mL-20ug/mL specific antigen PGRMC1, and the specific antigen PGRMC1 is coated on solid phase
On reactive group bottom, after coating is good, extracts coating buffer out, throw off the chip, and stick another chip, make on second piece of chip
Pipeline it is vertical with the former pipeline placement direction of chip piece, intersecting angle is 90 °, and by itself and solid phase reaction substrates seal
Afterwards, it is passed through BSA confining liquid to microchannel, extracts confining liquid out after closing.
The preparation of specific antigen PGRMC1 coating solution: take the PGRMC1 that 1 μ L concentration is 0.358mg/mL that 17- is added
PGRMC1 coating buffer is prepared in the PBS buffer solution of 119uL, dilution PGRMC1 coating buffer concentration is 3ug/mL-20ug/mL, is mixed
It is even, the pH7.2-7.4 of PBS.
Solid phase reaction substrate of the present invention be polystyrene, dimethyl silicone polymer, polymethyl methacrylate one
Kind is a variety of, preferably polystyrene.
The Microfluidic Immunoassay Chip of 1 albumen of progesterone receptor membrane component in a kind of detection blood of the present invention
Using:
(1) 20uL object to be detected is added in the microchannel well of the Microfluidic Immunoassay Chip of Xiang Suoshu, is incubated for
20-60min is washed with the cleaning solution containing 0.05% Tween-20；
(2) the detection antibody 10uL of enzyme label is added into step (1) described microchannel, with containing 0.05% tween
Cleaning solution is washed；The goat anti-rabbit antibody of the preferred HRP label of the detection antibody of enzyme label；
(3) luminol chemiluminescence substrate 20uL is added into step (2) described microchannel, passes through chemiluminescence detection
Instrument is detected.
Coating substance used of the invention is specific antigen PGRMC1 albumen, can be coated in solid phase substrate, used anti-
Body is purchase or preparation antibody K1 corresponding with specific antigen PGRMC1 albumen, is avoided that nonspecific reaction, can specificity
Identification whole blood in PGRMC1, and be immunoreacted therewith.
Step (1) object to be detected of the present invention is the solution after the reaction of blood antibody K1 corresponding with antigen PGRMC1.
The blood that the present invention uses can quickly and accurately be diagnosed to be the concentration of PGRMC1 in human blood for whole blood or serum, to
Realize early diagnosing mammary cancer.
Step (1) the of the present invention incubation time is preferably 30min.Step (3) the of the present invention chemical luminescence for liquid is Shandong
Minot chemiluminescent substrate, the substrate can be by HRP enzymatics.The cleaning solution of washing of the present invention is to spit containing 0.05%
The cleaning solution of temperature -20, can reduce jamming pattern caused by microchannel non-specific adsorption using the cleaning solution.
As optimal technical scheme, the present invention the following steps are included:
(1) it the production of Microfluidic Immunoassay Chip: is formed after 7 duct chips are bonded with solid phase reaction substrate closed
Microchannel, 20uL is injected separately into Xiang Suoshu microchannel, and concentration is 17.9ug/mL specific antigen PGRMC1, the spy
Specific Antigen PGRMC1 is coated in substrate, after coating is good, is extracted coating buffer out, is thrown off the chip, and stick another core
Piece keeps pipeline on second piece of chip vertical with the former pipeline placement direction of chip piece, and by itself and solid phase reaction substrate
After sealing, it is passed through BSA confining liquid to microchannel, extracts confining liquid out after closing；
(2) each pipeline is added in the microchannel well into step (1) described Microfluidic Immunoassay Chip
20uL object to be detected is incubated for 30min, is washed with the cleaning solution containing 0.05% Tween-20, and each pipeline is added 20uL and washes
Liquid is washed, is then sucked out, is washed in triplicate；
(3) goat anti-rabbit antibody of HRP label is added into step (2) described microchannel, 20uL is added in each pipeline, uses
Containing 0.05% Tween-20 cleaning solution, 20uL is added in each pipeline, is then sucked out, cleaning solution in triplicate is washed；
(4) luminol chemiluminescence substrate is added into step (3) described microchannel, 20uL is added in each pipeline, leads to
Chemiluminescence detector is crossed to be detected.
The preparation of the specific antigen PGRMC1 coating buffer: the PGRMC1 that 1 μ L concentration is 0.358mg/mL is taken to be added
PGRMC1 coating is prepared in PBS (pH7.2-7.4) buffer solution of 17-119uL, dilutes PGRMC1 peridium concentration 3ug/mL-
20ug/mL is mixed.
The preparation of the object to be detected: taking 1 μ L concentration is the 3%BSA confining liquid that 500uL is added in 1.35mg/mL antibody
In, it is diluted to 2.7ug/mL, blood or serum is added, reacts 60min.
Illustrate that term used herein is defined as follows:
Term " PBS " refers to: phosphate buffered saline solution.
Term " BSA " refers to: bovine serum albumin(BSA).
Term " HRP " refers to: horseradish peroxidase.
Term " PGRMC1 " refers to: (the progesterone receptor membrane of progesterone receptor membrane component 1
components1)
PBS buffer solution (0.01mol/L) is prepared: 4.0g NaCl, 0.1g KH2PO4, 1.48g Na2HPO4·12H2O,
It is dissolved in 500mL distilled water, adjusts pH to 7.4
PBST cleaning solution (concentration 0.05%) is prepared: 0.05%Tween-20 is added in 0.01mol/L PBS: by 50uL
Tween-20 dissolves in the 0.01mol/L phosphate buffer of 100mL, and concussion mixes.
Testing principle of the invention is:
In the Microfluidic Immunoassay Chip prepared, object to be detected (object to be detected is added into microchannel well
For solution after the reaction of whole blood or serum antibody K1 corresponding with antigen PGRMC1), the antigen PGRMC1 in blood or serum is
Through terminating with excessive antibody response, remaining antibody is then integrated on envelope antigen, so that antigen-antibody complex is formed,
Then the antibody of enzyme label is added into microfluidic channel.To form Ag-Ab-enzyme label antibody conjugates, then lead to
Enter luminescent solution and react and generate luminous signal with enzyme generation catalysis, signal will be detected by chemiluminescent analyzer and provide number
Value.
Compared with prior art, the present invention at least has the advantages that
1, the present invention by Microfluidic Immunoassay Chip and selects whole blood or serum as sample, by disposably grasping
Make the content for detecting PGRMC1 in multiple samples, few, high-throughput detection, high specificity, high sensitivity, valence with sample size
Lattice are cheap, read the advantages that result quantifies and is intuitive；
2, the present invention shortens antigen set time, antigen-antibody reaction time；And it will be entirely immunoreacted time shortening,
It is fixed including antigen, the reaction of antibody response, secondary antibody, realizes quick detection；
3, the present invention does not need complicated instrument and equipment, does not need the micro-judgment of experimenter yet, avoids different behaviour
Make the subjective factor of personnel, the coincidence rate of testing result is higher, is suitable for clinical early diagnosis.
Detailed description of the invention
Fig. 1 is the result schematic diagram that the present invention detects several samples simultaneously
Fig. 2 is the flow chart of micro-fluidic chip immune detection of the present invention
Fig. 3 is antigen concentration and chemiluminescence intensity relation curve
Fig. 4 is the logarithm of the concentration of antigen and the standard curve of chemiluminescence intensity
Specific embodiment
Reagent instrument and equipment source used in embodiment:
(1) 1 albumen of progesterone receptor membrane component: biotechnology (Nanjing) Co., Ltd, moral Thailand or Santa Cruz biotechnology
Co., Ltd；
(2) PGRMC1 protein antibodies: biotechnology (Nanjing) Co., Ltd, moral Thailand or Genetimes
Technology.Inc.
Biotechnology (group) company, lucky Thailand or Santa Cruz Bioisystech Co., Ltd；
(3) goat anti-rabbit antibody: Proteintech or Santa Cruz Bioisystech Co., Ltd；
(4) chip (PS) substrate: corning
(5) PDMS chip matrix, curing agent: DOW CORNING 184
(6) high temperature drying case: the permanent Science and Technology Ltd. in Shanghai one
(7) PBS: Beijing Chemical Co., Ltd. or Beijing Putin's health Li Co., Ltd
(8) chemical luminescence for liquid: milipore (dealer Beijing Putin's health Li Co., Ltd)
(9) Chemiluminescence Apparatus: State Nanometer Science Center develops or Mai Ruier experimental facilities (Shanghai) Co., Ltd.
Embodiment 1
(1) closing the production of Microfluidic Immunoassay Chip: is formed after seven pipeline chips are bonded with solid phase reaction substrate
7 microchannels, then take 1 μ L concentration be 0.358mg/mL PGRMC1 be added 19uL PBS (pH7.2-7.4) buffering it is molten
PGRMC1 is prepared in liquid and is coated with solution, and dilution PGRMC1 coating solution concentration is 17.9ug/mL, is mixed；PGRMC1 after diluting
Coating solution is passed through in Micro-flow pipe.(BSA of 0.03g is weighed, so with the BSA lock solution of PBS buffer preparation 3%
Afterwards plus PBS to 10mL).Chip piece is thrown off, after air-drying, second piece of chip is sticked in substrate, is made on second piece of chip
Pipeline is vertical with the former pipeline placement direction of chip piece, sealing；Then 20 μ L are passed through to each pipeline with pipettor
BSA confining liquid, room temperature 30min；It extracts confining liquid out, after dry, is put into hermetic bag, 4 DEG C of preservations obtain Microfluidic Immunoassay
Chip.
(2) object to be detected prepares: taking 1 μ L concentration is that 1.35mg/mL antibody is added in the 3%BSA confining liquid of 500uL, dilute
It is interpreted into 2.7ug/mL, blood or serum is added, reacts a hour.
(3) liquid to be detected is added in the microchannel well into step (1) described Microfluidic Immunoassay Chip, often
20uL is added in a pipeline, is incubated for 60min, and with the cleaning solution containing 0.05% tween, every hole is added 20uL, is then sucked out, and repeats
Three times, it is washed；
(4) goat anti-rabbit antibody of HRP label is added into step (3) described microchannel, with containing 0.05% tween
Cleaning solution is washed；
(5) luminol chemiluminescence substrate 20uL is added into step (4) described microchannel, passes through chemiluminescence detection
Instrument is detected.
(6) it is put into Chemiluminescence Apparatus, carries out data analysis using analysis software；As a result judge: testing result such as Fig. 1 institute
Show, after being passed through sample, luminous signal is generated on chip, the cutoff numerical value of luminous signal is read through chemiluminescent analyzer.
PGRMC1 content in 1 Whole Blood of Healthy of table
| Number | C1 | C2 | C3 | C4 | C5 |
| Concentration (pg/mL) | 22.46±6 | 22.69±5.2 | 25.40±4.8 | 21.18±5.6 | 18.82±4.2 |
Embodiment 2
(1) it the production of Microfluidic Immunoassay Chip: is formed after 7 pipeline chips are bonded with solid phase reaction substrate closed
Then 7 microchannels take the PGRMC1 that 1 μ L concentration is 0.358mg/mL that PBS (pH7.2-7.4) buffer solution of 39uL is added
Middle preparation PGRMC1 is coated with solution, and dilution PGRMC1 coating solution concentration is 8.95ug/mL, mixes；PGRMC1 packet after diluting
It is passed through in Micro-flow pipe by solution.(BSA of 0.03g is weighed, then with the BSA lock solution of PBS buffer preparation 3%
Add PBS to 10mL).Chip piece is thrown off, after air-drying, second piece of chip is sticked in substrate, makes the pipe on second piece of chip
Road is vertical with the former pipeline placement direction of chip piece, sealing；Then 20 μ L BSA are passed through to each pipeline with pipettor
Confining liquid, room temperature 30min；It extracts confining liquid out, after dry, is put into hermetic bag, 4 DEG C of preservations obtain Microfluidic Immunoassay core
Piece.
(2) object to be detected prepares: taking 1 μ L concentration is that 1.35mg/mL antibody is added in the 3%BSA confining liquid of 500uL, dilute
It is interpreted into 2.7ug/mL, the blood or serum for being added but detecting react a hour.
(3) liquid to be detected is added in the microchannel well into step (1) described Microfluidic Immunoassay Chip, often
20uL is added in a pipeline, is incubated for 60min, and with the cleaning solution containing 0.05% tween, every hole is added 20uL, is then sucked out, and repeats
Three times, it is washed；
(4) goat anti-rabbit antibody of HRP label is added into step (3) described microchannel, with containing 0.05% tween
Cleaning solution is washed；
(5) luminol chemiluminescence substrate 20uL is added into step (4) described microchannel, passes through chemiluminescence detection
Instrument is detected.
(6) luminous signal is generated on chip, and the cutoff numerical value of luminous signal is read through chemiluminescent analyzer.
PGRMC1 content in 2 Whole Blood of Healthy of table
Embodiment 3
(1) it the production of Microfluidic Immunoassay Chip: is formed after 7 pipeline chips are bonded with solid phase reaction substrate closed
Then 7 microchannels take the PGRMC1 that 1 μ L concentration is 0.358mg/mL that PBS (pH7.2-7.4) buffer solution of 79uL is added
Middle preparation PGRMC1 is coated with solution, and dilution PGRMC1 coating solution concentration is 17.9ug/mL, mixes；PGRMC1 packet after diluting
It is passed through in Micro-flow pipe by solution.(BSA of 0.03g is weighed, then with the BSA lock solution of PBS buffer preparation 3%
Add PBS to 10mL).Chip piece is thrown off, after air-drying, second piece of chip is sticked in substrate, makes the pipe on second piece of chip
Road is vertical with the former pipeline placement direction of chip piece, sealing；Then 20 μ L BSA are passed through to each pipeline with pipettor
Confining liquid, room temperature 30min；It extracts confining liquid out, after dry, is put into hermetic bag, 4 DEG C of preservations obtain Microfluidic Immunoassay core
Piece.
(2) object to be detected prepares: taking 1 μ L concentration is that 1.35mg/mL antibody is added in the 3%BSA confining liquid of 500uL, dilute
It is interpreted into 2.7ug/mL, blood or serum is added, reacts a hour.
(3) liquid to be detected is added in the microchannel well into step (1) described Microfluidic Immunoassay Chip, often
20uL is added in a pipeline, is incubated for 60min, and with the cleaning solution containing 0.05% tween, every hole is added 20uL, is then sucked out, and repeats
Three times, it is washed；
(4) goat anti-rabbit antibody of HRP label is added into step (3) described microchannel, with containing 0.05% tween
Cleaning solution is washed；
(5) luminol chemiluminescence substrate 20uL is added into step (4) described microchannel, passes through chemiluminescence detection
Instrument is detected.
(6) luminous signal is generated on chip, and the cutoff numerical value of luminous signal is read through chemiluminescent analyzer.
PGRMC1 content in 3 patients blood of table
Embodiment 4
(1) it the production of Microfluidic Immunoassay Chip: is formed after 7 pipeline chips are bonded with solid phase reaction substrate closed
Then 7 microchannels take the PGRMC1 that 1 μ L concentration is 0.358mg/mL that PBS (pH7.2-7.4) buffer solution of 79uL is added
Middle preparation PGRMC1 is coated with solution, and dilution PGRMC1 coating solution concentration is 17.9ug/mL, mixes；PGRMC1 is molten after diluting
Liquid is passed through in Micro-flow pipe.Then plus PBS (BSA of 0.03g is weighed, with the BSA lock solution of PBS buffer preparation 3%
To 10mL).Chip piece is thrown off, after air-drying, second piece of chip is sticked in substrate, makes pipeline and original on second piece of chip
The pipeline placement direction of chip piece is vertical, sealing；Then 20 μ L BSA confining liquids are passed through to each pipeline with pipettor,
Room temperature 30min；It extracts confining liquid out, after dry, is put into hermetic bag, 4 DEG C of preservations obtain Microfluidic Immunoassay Chip.
(2) object to be detected prepares: taking 1 μ L concentration is that 1.35mg/mL antibody is added in the 3%BSA confining liquid of 500uL, dilute
It is interpreted into 2.7ug/mL, blood or serum is added, reacts a hour.
(3) liquid to be detected is added in the microchannel well into step (1) described Microfluidic Immunoassay Chip, often
20uL is added in a pipeline, is incubated for 60min, and with the cleaning solution containing 0.05% tween, every hole is added 20uL, is then sucked out, and repeats
Three times, it is washed；
(4) goat anti-rabbit antibody of HRP label is added into step (3) described microchannel, with containing 0.05% tween
Cleaning solution is washed；
(5) luminol chemiluminescence substrate 20uL is added into step (4) described microchannel, passes through chemiluminescence detection
Instrument is detected.
(6) luminous signal is generated on chip, and the cutoff numerical value of luminous signal is read through chemiluminescent analyzer.
PGRMC1 content in 4 patients blood of table
Embodiment 5
(1) it the production of Microfluidic Immunoassay Chip: is formed after 7 pipeline chips are bonded with solid phase reaction substrate closed
Then 7 microchannels take the PGRMC1 that 1 μ L concentration is 0.358mg/mL that PBS (pH7.2-7.4) dilute solution of 79uL is added
Middle preparation PGRMC1 is coated with solution, and dilution PGRMC1 coating solution concentration is 17.9ug/mL；PGRMC1 solution after dilution is passed through
In Micro-flow pipe.With the BSA lock solution of PBS buffer preparation 3% (weigh the BSA of 0.03g, then plus PBS extremely
10mL).Chip piece is thrown off, after air-drying, second piece of chip is sticked in substrate, makes pipeline on second piece of chip and former the
The pipeline placement direction of chip piece is vertical, sealing；Then 20 μ L BSA confining liquids, room are passed through to each pipeline with pipettor
Warm 30min；It extracts confining liquid out, after dry, is put into hermetic bag, 4 DEG C of preservations obtain Microfluidic Immunoassay Chip.
(2) object to be detected prepares: taking 1 μ L concentration is that 1.35mg/mL antibody is added in the 3%BSA confining liquid of 500uL, dilute
It is interpreted into 2.7ug/mL, blood or serum is added, reacts a hour.
(3) liquid to be detected is added in the microchannel well into step (1) described Microfluidic Immunoassay Chip, often
20uL is added in a pipeline, is incubated for 60min, and with the cleaning solution containing 0.05% tween, every hole is added 20uL, is then sucked out, and repeats
Three times, it is washed；
(4) goat anti-rabbit antibody of HRP label is added into step (3) described microchannel, with containing 0.05% tween
Cleaning solution is washed；
(5) luminol chemiluminescence substrate 20uL is added into step (4) described microchannel, passes through chemiluminescence detection
Instrument is detected.
(6) luminous signal is generated on chip, and the cutoff numerical value of luminous signal is read through chemiluminescent analyzer.
PGRMC1 content in 5 patients blood of table
Embodiment 6
(1) it the production of Microfluidic Immunoassay Chip: is formed after 3 pipeline chips are bonded with solid phase reaction substrate closed
Then 3 microchannels take the PGRMC1 that 1 μ L concentration is 0.358mg/mL that PBS (pH7.2-7.4) buffer solution of 79uL is added
Middle preparation PGRMC1 is coated with solution, and dilution PGRMC1 coating solution concentration is 17.9ug/mL；PGRMC1 solution after dilution is passed through
In Micro-flow pipe.With the BSA lock solution of PBS buffer preparation 3% (weigh the BSA of 0.03g, then plus PBS extremely
10mL).Chip piece is thrown off, after air-drying, second piece of chip is sticked in substrate, makes pipeline on second piece of chip and former the
The pipeline placement direction of chip piece is vertical, sealing；Then 20 μ L BSA confining liquids, room are passed through to each pipeline with pipettor
Warm 30min；It extracts confining liquid out, after dry, is put into hermetic bag, 4 DEG C of preservations obtain Microfluidic Immunoassay Chip.
(2) object to be detected prepares: taking 1 μ L concentration is that 1.35mg/mL antibody is added in the 3%BSA confining liquid of 500uL, dilute
It is interpreted into 2.7ug/mL, blood or serum is added, reacts a hour.
(3) liquid to be detected is added in the microchannel well into step (1) described Microfluidic Immunoassay Chip, often
20uL is added in a pipeline, is incubated for 60min, and with the cleaning solution containing 0.05% tween, every hole is added 20uL, is then sucked out, and repeats
Three times, it is washed；
(4) goat anti-rabbit antibody of HRP label is added into step (3) described microchannel, with containing 0.05% tween
Cleaning solution is washed；
(5) luminol chemiluminescence substrate 20uL is added into step (4) described microchannel, passes through chemiluminescence detection
Instrument is detected.
(6) luminous signal is generated on chip, and the cutoff numerical value of luminous signal is read through chemiluminescent analyzer.
PGRMC1 content in 6 patients blood of table
Embodiment 7
(1) it the production of Microfluidic Immunoassay Chip: is formed after 9 pipeline chips are bonded with solid phase reaction substrate closed
Then 9 microchannels take the PGRMC1 that 1 μ L concentration is 0.358mg/mL that PBS (pH7.2-7.4) buffer solution of 79uL is added
Middle preparation PGRMC1 is coated with solution, and dilution PGRMC1 coating solution concentration is 17.9ug/mL；PGRMC1 solution after dilution is passed through
In Micro-flow pipe.With the BSA lock solution of PBS buffer preparation 3% (weigh the BSA of 0.03g, then plus PBS extremely
10mL).Chip piece is thrown off, after air-drying, second piece of chip is sticked in substrate, makes pipeline on second piece of chip and former the
The pipeline placement direction of chip piece is vertical, sealing；Then 20 μ L BSA confining liquids, room are passed through to each pipeline with pipettor
Warm 30min；It extracts confining liquid out, after dry, is put into hermetic bag, 4 DEG C of preservations obtain Microfluidic Immunoassay Chip.
(2) object to be detected prepares: taking 1 μ L concentration is that 1.35mg/mL antibody is added in the 3%BSA confining liquid of 500uL, dilute
It is interpreted into 2.7ug/mL, blood or serum is added, reacts a hour.
(3) liquid to be detected is added in the microchannel well into step (1) described Microfluidic Immunoassay Chip, often
20uL is added in a pipeline, is incubated for 60min, and with the cleaning solution containing 0.05% tween, every hole is added 20uL, is then sucked out, and repeats
Three times, it is washed；
(4) goat anti-rabbit antibody of HRP label is added into step (3) described microchannel, with containing 0.05% tween
Cleaning solution is washed；
(5) luminol chemiluminescence substrate 20uL is added into step (4) described microchannel, passes through chemiluminescence detection
Instrument is detected.
(6) luminous signal is generated on chip, and the cutoff numerical value of luminous signal is read through chemiluminescent analyzer.
PGRMC1 content in 7 patients blood of table
Embodiment 8
(1) closed 21 the production of Microfluidic Immunoassay Chip: are formed after chip is bonded with solid phase reaction substrate
Then microchannel takes the PGRMC1 that 1 μ L concentration is 0.358mg/mL to be added in PBS (pH7.2-7.4) buffer solution of 79uL
It prepares PGRMC1 and is coated with solution, dilution PGRMC1 coating solution concentration is 17.9ug/mL；PGRMC1 solution after dilution is passed through micro-
In flow control pipeline.Then plus PBS to 10mL) (BSA of 0.03g is weighed, with the BSA lock solution of PBS buffer preparation 3%.
Chip piece is thrown off, after air-drying, second piece of chip is sticked in substrate, makes pipeline and former first piece of core on second piece of chip
The pipeline placement direction of piece is vertical, sealing；Then 20 μ L BSA confining liquids, room temperature are passed through to each pipeline with pipettor
30min；It extracts confining liquid out, after dry, is put into hermetic bag, 4 DEG C of preservations obtain Microfluidic Immunoassay Chip.
(2) object to be detected prepares: taking 1 μ L concentration is that 1.35mg/mL antibody is added in the 3%BSA confining liquid of 500uL, dilute
It is interpreted into 2.7ug/mL, blood or serum is added, reacts a hour.
(3) liquid to be detected is added in the microchannel well into step (1) described Microfluidic Immunoassay Chip, often
20uL is added in a pipeline, is incubated for 30min, and with the cleaning solution containing 0.05% tween, every hole is added 20uL, is then sucked out, and repeats
Three times, it is washed；
(4) goat anti-rabbit antibody of HRP label is added into step (3) described microchannel, with containing 0.05% tween
Cleaning solution is washed；
(5) luminol chemiluminescence substrate 20uL is added into step (4) described microchannel, passes through chemiluminescence detection
Instrument is detected.
(6) luminous signal is generated on chip, and the cutoff numerical value of luminous signal is read through chemiluminescent analyzer.
Embodiment 9
(1) closed 15 the production of Microfluidic Immunoassay Chip: are formed after chip is bonded with solid phase reaction substrate
Then microchannel takes the PGRMC1 that 1 μ L concentration is 0.358mg/mL to be added in PBS (pH7.2-7.4) buffer solution of 79uL
It prepares PGRMC1 and is coated with solution, dilution PGRMC1 coating solution concentration is 17.9ug/mL；PGRMC1 solution after dilution is passed through micro-
In flow control pipeline.Then plus PBS to 10mL) (BSA of 0.03g is weighed, with the BSA lock solution of PBS buffer preparation 3%.
Chip piece is thrown off, after air-drying, second piece of chip is sticked in substrate, makes pipeline and former first piece of core on second piece of chip
The pipeline placement direction of piece is vertical, sealing；Then 20 μ L BSA confining liquids, room temperature are passed through to each pipeline with pipettor
30min；It extracts confining liquid out, after dry, is put into hermetic bag, 4 DEG C of preservations obtain Microfluidic Immunoassay Chip.
(2) object to be detected prepares: taking 1 μ L concentration is that 1.35mg/mL antibody is added in the 3%BSA confining liquid of 500uL, dilute
It is interpreted into 2.7ug/mL, blood or serum is added, reacts a hour.
(3) liquid to be detected is added in the microchannel well into step (1) described Microfluidic Immunoassay Chip, often
20uL is added in a pipeline, is incubated for 40min, and with the cleaning solution containing 0.05% tween, every hole is added 20uL, is then sucked out, and repeats
Three times, it is washed；
(4) goat anti-rabbit antibody of HRP label is added into step (3) described microchannel, with containing 0.05% tween
Cleaning solution is washed；
(5) luminol chemiluminescence substrate 20uL is added into step (4) described microchannel, passes through chemiluminescence detection
Instrument is detected.
(6) luminous signal is generated on chip, and the cutoff numerical value of luminous signal is read through chemiluminescent analyzer.
Embodiment 10
(1) closed 15 the production of Microfluidic Immunoassay Chip: are formed after chip is bonded with solid phase reaction substrate
The PGRMC1 solution of 3ug/mL is passed through in Micro-flow pipe by microchannel.It is molten with the BSA closing of PBS buffer preparation 3%
Then plus PBS to 10mL) liquid (weighs the BSA of 0.03g,.Chip piece is thrown off, after air-drying, second piece of core is sticked in substrate
Piece keeps the pipeline on second piece of chip vertical with the former pipeline placement direction of chip piece, sealing；Then with pipettor to every
A pipeline is passed through 20 μ L BSA confining liquids, room temperature 30min；It extracts confining liquid out, after dry, is put into hermetic bag, 4 DEG C save, i.e.,
Microfluidic Immunoassay Chip is made.
(2) object to be detected prepares: taking 1 μ L concentration is that 1.35mg/mL antibody is added in the 3%BSA confining liquid of 500uL, dilute
It is interpreted into 2.7ug/mL, blood or serum is added, reacts a hour.
(3) liquid to be detected is added in the microchannel well into step (1) described Microfluidic Immunoassay Chip, often
20uL is added in a pipeline, is incubated for 60min, and with the cleaning solution containing 0.05% tween, every hole is added 20uL, is then sucked out, and repeats
Three times, it is washed；
(4) goat anti-rabbit antibody of HRP label is added into step (3) described microchannel, with containing 0.05% tween
Cleaning solution is washed；
(5) luminol chemiluminescence substrate 20uL is added into step (4) described microchannel, passes through chemiluminescence detection
Instrument is detected.
(6) luminous signal is generated on chip, and the cutoff numerical value of luminous signal is read through chemiluminescent analyzer.
Embodiment 11
(1) closed 12 the production of Microfluidic Immunoassay Chip: are formed after chip is bonded with solid phase reaction substrate
The PGRMC1 solution of 20ug/mL is passed through in Micro-flow pipe by microchannel.It is closed with the BSA of PBS buffer preparation 3%
Then plus PBS to 10mL) solution (weighs the BSA of 0.03g,.Chip piece is thrown off, after air-drying, second piece is sticked in substrate
Chip keeps the pipeline on second piece of chip vertical with the former pipeline placement direction of chip piece, sealing；Then with pipettor to
Each pipeline is passed through 20 μ L BSA confining liquids, room temperature 30min；It extracts confining liquid out, after dry, is put into hermetic bag, 4 DEG C save,
Obtain Microfluidic Immunoassay Chip.
(2) object to be detected prepares: taking 1 μ L concentration is that 1.35mg/mL antibody is added in the 3%BSA confining liquid of 500uL, dilute
It is interpreted into 2.7ug/mL, blood or serum is added, reacts a hour.
(3) liquid to be detected is added in the microchannel well into step (1) described Microfluidic Immunoassay Chip, often
20uL is added in a pipeline, is incubated for 60min, and with the cleaning solution containing 0.05% tween, every hole is added 20uL, is then sucked out, and repeats
Three times, it is washed；
(4) goat anti-rabbit antibody of HRP label is added into step (3) described microchannel, with containing 0.05% tween
Cleaning solution is washed；
(5) luminol chemiluminescence substrate 20uL is added into step (4) described microchannel, passes through chemiluminescence detection
Instrument is detected.
(6) luminous signal is generated on chip, and the cutoff numerical value of luminous signal is read through chemiluminescent analyzer.
Embodiment 12
(1) closed 12 the production of Microfluidic Immunoassay Chip: are formed after chip is bonded with solid phase reaction substrate
The PGRMC1 solution of 10ug/mL is passed through in Micro-flow pipe by microchannel.It is closed with the BSA of PBS buffer preparation 3%
Then plus PBS to 10mL) solution (weighs the BSA of 0.03g,.Chip piece is thrown off, after air-drying, second piece is sticked in substrate
Chip keeps the pipeline on second piece of chip vertical with the former pipeline placement direction of chip piece, sealing；Then with pipettor to
Each pipeline is passed through 20 μ L BSA confining liquids, room temperature 30min；It extracts confining liquid out, after dry, is put into hermetic bag, 4 DEG C save,
Obtain Microfluidic Immunoassay Chip.
(2) object to be detected prepares: taking 1 μ L concentration is that 1.35mg/mL antibody is added in the 3%BSA confining liquid of 500uL, dilute
It is interpreted into 2.7ug/mL, blood or serum is added, reacts a hour.
(3) liquid to be detected is added in the microchannel well into step (1) described Microfluidic Immunoassay Chip, often
20uL is added in a pipeline, is incubated for 60min, and with the cleaning solution containing 0.05% tween, every hole is added 20uL, is then sucked out, and repeats
Three times, it is washed；
(4) goat anti-rabbit antibody of HRP label is added into step (3) described microchannel, with containing 0.05% tween
Cleaning solution is washed；
(5) luminol chemiluminescence substrate 20uL is added into step (4) described microchannel, passes through chemiluminescence detection
Instrument is detected.
(6) luminous signal is generated on chip, and the cutoff numerical value of luminous signal is read through chemiluminescent analyzer.
Although above having used general explanation, specific embodiment and test, the present invention is made to retouch in detail
It states, but on the basis of the present invention, it can be modified or is improved, this is aobvious and easy to those skilled in the art
See.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to claimed
Range.
Claims (8)
1. the Microfluidic Immunoassay Chip of 1 albumen of progesterone receptor membrane component in a kind of detection blood, it is characterised in that: this is micro-
Flow control Immunoassay Chip is by chip and solid phase reaction substrate and the specific antigen PGRMC1 being coated in solid phase reaction substrate
Composition；Chip is the laminated structure with parallel microchannel, and the specific antigen PGRMC1 in solid phase reaction substrate is parallel
Streak-like planar structure is mutually perpendicular to the chip microchannel, and intersecting angle is 90 °；The chip be manually overmolded or
The laminated structure with parallel microchannel being molded；The microchannel length is 3-5cm, the microchannel diameter
500-600 μm 500-600 μm of height, the spacing of the microchannel is 1-3mm；
The solid phase reaction substrate be selected from polystyrene, dimethyl silicone polymer, polymethyl methacrylate it is one or more.
The specific antigen PGRMC1 being coated in solid phase reaction substrate be parallel streak-like planar structure, length 3-5cm,
Spacing is 1-3mm, is mutually perpendicular to the chip microchannel, and intersecting angle is 90 °.
2. the Microfluidic Immunoassay of 1 albumen of progesterone receptor membrane component in a kind of detection blood according to claim 1
Chip, it is characterised in that: the chip has the parallel microchannel of 3-21 item.
3. the Microfluidic Immunoassay Chip preparation method of 1 albumen of progesterone receptor membrane component, feature in a kind of detection blood
It is:
Closed microchannel is formed after chip is bonded with solid phase reaction substrate, is injected separately into Xiang Suoshu microchannel
20uL, concentration range are 3ug/mL-20ug/mL specific antigen PGRMC1, and the specific antigen PGRMC1 is coated on solid phase
On reactive group bottom, after coating is good, extracts coating buffer out, throw off the chip, and stick another chip, make on second piece of chip
Pipeline it is vertical with the former pipeline placement direction of chip piece, intersecting angle is 90 °, and by itself and solid phase reaction substrates seal
Afterwards, it is passed through BSA confining liquid to microchannel, extracts confining liquid out after closing.
4. the Microfluidic Immunoassay of 1 albumen of progesterone receptor membrane component in a kind of detection blood according to claim 3
Chip preparation method, it is characterised in that: the preparation of the specific antigen PGRMC1 coating buffer: the 1 μ L concentration is taken to be
The PGRMC1 of 0.358mg/mL, which is added in the PBS buffer solution of 17-119uL, prepares PGRMC1 coating buffer, dilution PGRMC1 coating
Liquid concentration is 3ug/mL-20ug/mL, is mixed, the pH7.2-7.4 of PBS.
5. the application of the Microfluidic Immunoassay Chip of 1 albumen of progesterone receptor membrane component, feature exist in a kind of detection blood
In:
(1) 20uL object to be detected is added in the microchannel well of the Microfluidic Immunoassay Chip of Xiang Suoshu, is incubated for 20-
60min is washed with the cleaning solution containing 0.05% Tween-20；
(2) the detection antibody 10uL of enzyme label is added into step (1) described microchannel, with the washing containing 0.05% tween
Liquid is washed；
(3) into step (2) described microchannel be added luminol chemiluminescence substrate 20uL, by chemiluminescence detector into
Row detection.
6. the Microfluidic Immunoassay of 1 albumen of progesterone receptor membrane component in a kind of detection blood according to claim 5
The application of chip, it is characterised in that: the detection antibody of the enzyme label is the goat anti-rabbit antibody of HRP label.
7. the Microfluidic Immunoassay of 1 albumen of progesterone receptor membrane component in a kind of detection blood according to claim 5
The application of chip, it is characterised in that: the object to be detected is the solution after blood antibody K1 reaction corresponding with antigen PGRMC1.
8. the Microfluidic Immunoassay of 1 albumen of progesterone receptor membrane component in a kind of detection blood according to claim 5
The application of chip, it is characterised in that: the preparation of the object to be detected: taking 1 μ L concentration is 1.35mg/mL antibody K1, is added
In the 3%BSA confining liquid of 500uL, it is diluted to 2.7ug/mL, it is isometric that blood or serum is added, react 20-60min.
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